Virulent M. tuberculosis can induce life-long immunity against reinfection in individuals who develop the latent form of this disease. The resulting immunity is substantially better than that usually achieved in BCG vaccinated people. Virulent M. tb H37Rv has been attenuated by a targeted mutagenesis of the LeuD or LysA genes or the NAD gene and the resulting auxotrophs injected into C57BL/6 mice to assess their survival in the lung and spleen as a measure of residual virulence and potential immunogenicity. The mice were infected 6-12 weeks later with a small aerogenic inoculum of M. tb Erdman and the growth in the lung was compared with that for a group of BCG controls. The auxotrophic H37RvLeu- mutant induced high levels of anti-tuberculous resistance but virulence tests carried out on this auxotroph indicated an unacceptable level of reversion to the wild type and further studies with this auxotroph were discontinued. Tests with lysine auxotrophs of BCG Pasteur and M. tb H37Rv have been carried out in C57BL/6 mice. Both auxotrophs failed to multiply in vitro in the absence of an extrinsic source of lysine and were unable to grow or survive in intravenously vaccinated mice. They were unable to induce detectable anti-tuberculous resistance against an aerogenic Erdman challenge administered 3 months later. However, mice which received 2 or 3 doses of the lysine auxotroph expressed enhanced resistance to challenge, approaching that observed in the BCG vaccinated controls. These experiments are continuing with other auotrophic strains of H37Rv and Erdman supplied by Dr. Jacobs for testing for virulence and immunogenicity.